基于不同引物的16S rRNA序列分析法对SBP腹水细菌的鉴定及抗感染疗效评价的意义

Clinical application of 16S rRNA sequencing analysis for diagnosis of spontaneous bacterial peritonitis and evaluation of antibiotic efficacy

目的:探讨联合单独或联合应用2对不同引物的16S r RNA序列分析法,能否提高自发性细菌性腹膜炎(spontaneous bacterial peritonitis,SBP)腹水细菌检测的敏感性及准确性,并且通过其检测细菌的结果来评价抗感染治疗效果.方法:收集77例肝硬化失代偿期合并腹水的标本,其中SBP 61例,非SBP 16例,应用2对不同引物(MSQ-F/MSQ-R与27F/1492R)的组合,对腹水细菌分别进行16S r RNA序列分析,对腹水进行常规化验、细菌培养及生化方法鉴定细菌,分析单独或联合应用2对不同引物的16S r RNA序列分析法在鉴定SBP腹水细菌中的应用价值,并对腹水细菌状态进行动态评价.结果:与腹水细菌培养、生化鉴定的细菌结果相比,16S r RNA序列分析法鉴定细菌的结果与之的符合率:单用引物MSQ-F/M S Q-R:属的符合率73.33%(11/15),种的符合率66.67%(10/15);单用27F/1492R引物:属的符合率80.00%(12/15),种的符合率73.3%(11/15);引物MSQ-F/MSQ-R联合27F/1492R:属的符合率86.67%(13/15),种的鉴定的符合率80.00%(12/15),应用2对引物的组合进行16S r RNA序列分析鉴定细菌,可以得到更高的准确率.5例SBP患者(腹水细菌培养阳性组2例,临床感染组3例)经抗感染治疗后,腹水细菌培养阴性,但临床感染控制不理想的患者,16S r RNA序列分析法检测腹水细菌仍为阳性.结论:联合应用2对不同引物的16S r RNA序列分析法,可以提高SBP腹水细菌的鉴定能力,并且可以动态应用于临床进行抗感染疗效评价.

AIM：To explore a new method of polymerase chain reaction（PCR） based on 16 S rRNA gene of bacteria with two different pairs of primers（used alone or in combination）and alignment sequencing,to improve the detection rate of bacteria in ascitic fluid and evaluate its clinical value in bacterial species identification and evaluation of antibiotic efficacy.METHODS：Blood and ascitic fluid samples were collected from 77 spontaneous bacterial peritonitis（SBP）（n= 61） cirrhotic patients with ascites.Bacterial culture of ascitic fluid was conducted and the results were used as the gold standard in this study.Bacterial DNA in ascitic fluid was detected by PCR,based on the 16 S rRNA gene,with two different pairs primers（MSQ-F/MSQ-R,27F/1492R） and alignment sequencing assay.The results of bacterial species and positive rate between PCR method and bacterial culture of ascitic fluid were compared,and the results of bacterial species identified with different pair primers were also compared.RESULTS：For products amplified with primers MSQ-F/MSQ-R,the coincidence rate of bacterial genus identification between PCR assay and ascitic fluid culture was 73.33%（11/ 15）,and the coincidence rate of bacterial species identification was66.67（10/ 15）.For products amplified with primers27F/ 1492 R,the coincidence rate of bacterial genus identification between PCR assay and ascitic fluid culture was80.00%（12/15）,and the coincidence rate of bacterial species identification was 73.33%（11/ 15）.For products amplified with primers MSQ-F/MSQ-R combined with primers 27F/1492 R,the coincidence rate of bacterial genus identification between PCR assay and ascitic fluid culture was 86.67%（13/15）,and the coincidence rate of bacterial species identification was 80.00%（12/ 15）.In five patients with SBP whose ascitic fluid culture was negative,PCR reaction was positive,which was consistent with clinical characteristics.CONCLUSION：The method combining the two different pairs of primers for identifying bacteria based on the 16 S rRNA gene and alignment sequencing has better identification ability and can evaluate antibiotic efficacy dynamically.